Proteases (also known as proteinases or peptidases) are proteolytic enzymes that catalyze the cleavage of peptide bonds in other proteins. The effect of such cleavage on protein molecules is diverse. In some instances, proteolytic cleavage causes the cleaved protein to become inactive. In other instances, proteolytic cleavage causes a once inactive protein to become activate. In yet other instances, proteolytic cleavage is a mechanism whereby a single polypeptide precursor is cleaved into two or more individual polypeptides.
Proteases, such as, for example, the serine proteases, have been linked to apoptosis (programmed cell death), and are believed to be involved in the development and progression of numerous diseases and disorders, including Alzheimer's and Parkinson's diseases, AIDS and cancer.
Proteases serve to degrade invading organisms, antigen-antibody complexes and certain tissue proteins that are no longer necessary or useful to the organism. In a normally functioning organism, proteases are produced in a limited quantity and are regulated in part through the synthesis of protease inhibitors. A large number of naturally occurring protease inhibitors serve to control the endogenous proteases by limiting their reactions locally and temporally. In addition, the protease inhibitors may inhibit proteases introduced into the body by infective agents. Tissues that are particularly prone to proteolytic attack and infection, e.g., those of the respiratory tract, are rich in protease inhibitors. Protease inhibitors have also become popular as a new class of anti-HIV drugs.
One class of protease inhibitors is the stefins. Stefins, or Type 1 cystatins, belong to a large, evolutionarily conserved protein superfamily, the members of which inhibit the papain-like cysteine proteinases. The molecular cloning and chromosomal localization of three members of the murine stefin gene family have been reported (Genomics 15(3):507-14 (1993)). These genes, designated as mouse stefins 1, 2, and 3, were isolated on the basis of their relatively increased expression in moth-eaten viable compared to normal congenic mouse bone marrow cells. The open reading frames of the stefin cDNAs were found to encode proteins of approximately 11.5 kDa that show between 50 and 92% identity to sequences of stefins isolated from various other species. Data from Southern analysis suggested that the murine stefin gene family encompasses at least 6 and possibly 10-20 members, all of which appear to be clustered in the genome. Analysis of interspecific backcross mice indicated that the genes encoding the three mouse stefins all map to mouse chromosome 16, a localization that is consistent with the assignment of the human stefin A gene to a region of conserved homology between human chromosome 3q and the proximal region of mouse chromosome 16.
Pennachio et al. found that mice lacking cystatin B (stefin 2) as a result of targeted disruption of the gene develop myoclonic seizures and ataxia similar to the symptoms shown in EPM1 (Nat. Genet. 20(3):251-8 (1998)). The principal cytopathology appeared to be loss of cerebellar granule cells, which frequently displayed condensed nuclei, fragmented DNA, and other cellular changes characteristic of apoptosis. This mouse model of EPM1 was thought to provide evidence that cystatin B, as a noncaspase cysteine protease inhibitor, has a role in preventing cerebellar apoptosis. Loss-of-function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as Unverricht-Lundborg disease (EPM1). The primary cellular events and mechanisms underlying the disease are unknown.
Given the importance of protease inhibitors, particularly stefins, a clear need exists for identification and characterization of protease inhibitors which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.